Histamine/GABA neurons in the regulation of striatal signaling and brain arousal

The hypothalamus is one of the oldest brain structures and it is responsible for the regulation of vital homeostatic functions. A small population of the neurons containing neurotransmitters histamine and GABA, reside in the posterior hypothalamus. Activation of these histamine/GABA neurons promotes attentive wakefulness and vigilance state. In addition to its main role in supporting wake state, histamine released from histamine/GABA neurons is involved in controlling appetite, water intake and energy expenditure. Although the role of GABA released from the histamine/GABA neurons has been much less studied, it is suggested to provide tonic inhibition in the cortical and striatal regions. There are three major topics addressed in the current work. First, we aimed to further probe the hypothesis of the dual histamine/GABA neurotransmitter phenotype of the hypothalamic histaminergic neurons. We were especially interested in whether histamine/GABA neurons are able to release GABA into the synapse via the vesicular transport mechanism. Our second aim was to investigate the consequences of global histamine deficiency with a focus on the cortico-striatal system and brain dopamine-histamine interactions. Lastly, we studied the possible role of GABA released from histamine/GABA neurons in tonic extrasynaptic inhibition. It is well established that histaminergic neurons are also GABAergic, based on the presence of GABA producing enzyme - glutamic acid decarboxylase (GAD) and GABA itself in histaminergic neurons, as demonstrated by immunohistochemical methods. Contradictory findings have been reported on the presence of the vesicular GABA transporter (Vgat) in these neurons. We used double fluorescence in situ hybridization (dFISH) to simultaneously detect GABAergic markers (GAD67 or Vgat mRNA) with a marker for histaminergic neurons - histidine decarboxylase (Hdc) mRNA. We confirmed that histamine/GABA neurons express Vgat mRNA and are able to release GABA via a classical Vgat-dependent mechanism. Previous research has shown the interaction of histamine and dopamine systems at the level of the striatum and proposed involvement of these two systems in neuropsychiatric disorders such as Gilles de la Tourette syndrome. Using a mouse line lacking the histamine-synthesizing enzyme (Hdc KO mice), we investigated the role of histamine in the regulation of the striatal system and its interaction with dopamine at the striatal level. We measured striatal dopamine and its metabolites levels with high performance liquid chromatography (HPLC) at the baseline and after treatment with dopamine precursor l-3,4-dihydroxyphenylalanine (L-Dopa). By quantitative polymerase chain reaction (qPCR), we compared the levels of striatal prodynorphin and proenkephalin transcripts in Hdc WT and KO mice. The transcript level of prodynorphin and proenkephalin is tightly regulated by the activity of principal striatal neurons, medium spiny neurons (MSN) and various neurotransmitters such as dopamine and acetylcholine. Furthermore, we performed detailed analyses of exploratory open field behavior of Hdc KO mice and used a stereological approach to assess the morphology and cytoarchitecture of the corticostriatal system in these mice. We found that Hdc KO mice had increased dopamine turnover in the striatum and impaired expression of striatal prodynorphin and proenkephalin transcripts. We hypothesized that global deficiency of histamine leads to upregulation of the dopaminergic system in the striatum, which in turn leads to altered behavioral structure observed in the novel open field test. Impaired dynorphin/κ-opioid receptor inhibitory feedback on the dopaminergic terminals might be responsible for the increased striatal dopamine release. Finally, we provided new evidence suggesting that GABA released from the histamine/GABA neurons acts on the extrasynaptic δ subunit containing GABAA receptors and provides tonic inhibition. Pharmacological activation of the histamine/GABA neurons in the mice lacking GABAA δ subunit (Gabrd KO) led to a hypervigilant phenotype in these mice as was shown by EEG recordings. In conclusion, we showed that histamine together with dopamine regulates striatal circuits and that GABA released from the histamine/GABA neurons regulates brain arousal state at least partially through the extrasynaptic δ subunit containing GABAA receptors.